In the past year, our research program has focused on: 1) understanding the role of microRNA-155 (miR-155) on CD8+ T cell biology and anti-tumor function, 2) understanding the role of transcription factor c-Myb on CD8+ T cell biology and anti-tumor function and 3) developing a clinical trial using anti-CD19 CAR-transduced CD8+ memory stem cells (Tscm). Project 1: We found that tumor-specific CD8+ T cells constitutively expressing miR-155 displayed enhanced proliferation and anti-tumor function compared to cells transduced with a scramble microRNA. Interestingly, the increased functionality of miR-155 overexpressing T cells was minimized following adoptive transfer into irradiated or genetically lymphodepleted hosts, suggesting that miR-155 enhances T cell activity only under conditions of limited homeostatic cytokines availability. Consistently, the increased functionality of miR-155 overexpressing CD8+ T cells was virtually abrogated in mice deficient of the homeostatic cytokines, interleukin-7 and interleukin-15. We identified numerous miR-155 targets, such as the Akt inhibitor Ship1 and several negative regulators of Stat signaling including Socs1 and Ptpn2. Accordingly, miR-155 overexpressing T cells exhibited enhanced activity of Stat5 and Akt, two pivotal pathways downstream homeostatic cytokine signaling. Expression of constitutively active Stat5a recapitulated the survival advantages conferred by miR-155 while constitutive Akt activation promoted sustained effector functions, indicating that Stat5 and Akt play non-redundant and distinctive roles in driving miR-155-mediated numeric and functional advantages. To gain further insights on the biology behind miR-155 activity, we performed massively parallel RNA sequencing (RNA-Seq) and compared the gene expression profiles of miR-155-overexpressing and control CD8+ T cells. Gene Set Enrichment Analysis revealed that miR-155 overexpressing cells downregulated numerous genes that are silenced in embryonic stem cells by the Polycomb Repressive Complex 2 (PRC2) through trimethylation of H3K27. Genome-wide H3K4 and H3K27 trimethylation analysis indicated that miR-155 enhanced H3K27me3 at promoters of genes involved in T cell senescence. We are currently exploring the possibility that miR-155 might prevent terminal differentiation in T cells by targeting the Jumonji- and AT-rich epigenetic regulator Jarid2, an important regulator of PRC2 activity or indirectly affecting other PRC2 components. Project 2: We have started investigating the role of the transcription factor c-Myb in CD8+ T cell differentiation and memory formation. We found that c-Myb is preferentially expressed in a subset of long-lived memory T cells that we recently identified, called T memory stem cells. Using transgenic mice and gene engineering approaches we found that c-Myb is a critical regulator CD8+ T cell stemness. T cells lacking c-Myb have a propensity to undergo terminal differentiation and senescence whereas cells overexpressing this transcription factor preferentially form long-lived memory cells. We are currently working to elucidate the molecular mechanisms downstream c-Myb activity. We identified downstream transcription factors influenced by c-Myb activity and we are currently exploring whether manipulation of these genes can rescue c-Myb-deficient T cells. Project 3: We have recently described in mice and humans a new memory T cell population endowed with the stem cell-like attributes of self-renewal and multipotency (Gattinoni at al. Nature Med 2009 and 2011). These T cells, termed stem cell memory T (Tscm) cells were capable of reconstituting the full diversity of memory and effector T cell compartments on serial transplantation. Most importantly, Tscm cells displayed robust proliferative and survival capacities and eradicated large established tumors even when limited numbers of cells were transferred, a condition in which other memory T cell subsets had little or no impact. We wouldlike now to bring these exciting discoveries into the clinical arena to improve the outcome of current T-based immunotherapies. We have initiated a collaboration with intramural investigators (James Kochenderfer, NCI, David Stroncek, CC and Mario Roederer, NIAID) with the goal of developing a phase I dose-escalation trial using anti-CD19 CAR-modified Tscm cells for the treatment of patients with metastatic CD19+ B cell malignancies refractory to prior allogeneic stem transplantation or with advanced B-cell malignancies who have never undergone allogeneic stem transplantation. Together with the Department of Transfusion Medicine, CC, we have recently established a manufacturing procedure for the generation of anti-CD19 CAR-modified Tscm cells at clinical scale under GMP conditions. In vitro-generated CD19-CAR modified Tscm cells were phenotypically, functionally and transcriptomically equivalent to their naturally occurring counterpart. Compared with T cell products currently under clinical investigation, CD19-CAR modified Tscm cells exhibited enhanced metabolic fitness, persistence and anti-tumor activity against systemic acute lymphoblastic leukemia xenografts.